PROJECT SUMMARY Human papillomaviruses (HPVs) are highly prevalent pathogens causally associated with over 5% of all human cancers, including ~25% of head and neck squamous cell carcinoma (HNSCC). The incidence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has increased steeply in the last two decades, and will likely comprise the majority of all HNSCCs in the US and worldwide by 2020. With current therapies, patients must deal with the profound sequelae of treatment, which requires considerable support from health and social care systems. Therefore, novel therapies that decrease morbidity and increase cure rates are essential for patients with HPV-positive HNSCC (HPV+ HNSCC). To develop successful patient therapies for HNSCC, an in-depth understanding of how HPV suppresses the host immune response is urgently needed. Our recent studies have revealed that, while the proinflammatory chemokines are upregulated, a relatively novel chemokine CXCL14 is significantly downregulated in HPV+ HNSCC by the HPV oncoprotein E7-induced CXCL14 promoter hypermethylation. Restoration of CXCL14 expression in HPV+ mouse HNSCC cells suppresses tumor growth in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Mice with CXCL14 expression showed dramatically increased CD8+ T and natural killer (NK) cell populations in tumor tissues and tumor draining lymph nodes, and decreased immunosuppressive immune cells including myeloid- derived suppressor cells (MDSCs). Thus, we hypothesize that CXCL14 downregulation by HPV E7 drives HNSCC development by inducing MDSC infiltration into the TME and failing to elicit CD8+ T and NK cell responses; and that CXCL14 expression and immune cell infiltration in the TME correlate with clinical outcomes of HNSCC patients and boosts tumor suppression in combination with chemoradiation therapy (CRT). This hypothesis will be tested through 4 specific aims: 1) Determine whether CXCL14-induced CD8+ T and NK cells are necessary and sufficient for the antitumor immune response to HNSCC cells, using our immunocompetent syngeneic mouse models transplanted with engineered HPV+ mouse HNSCC cells. 2) Define the mechanisms by which the HPV oncoprotein E7 downregulates CXCL14 expression through promoter hypermethylation, using our promoter methylation reporter assay and HPV16 E7 mutants. 3) Assess the clinical correlation between CXCL14 expression, immune cell infiltration, and clinical outcomes of HNSCC patients, using clinical specimens from HNSCC patients for developing useful prognostic tools. 4) Define if chemoradiation therapy changes HPV E7 and CXCL14 expression in HPV+ HNSCC cells and how CXCL14 re-expression impacts tumor clearance during standard therapies, using in vitro and in vivo chemoradiation therapy in combination with Cxcl14 expression in our our immunocompetent mouse HNSCC model. This proposed study will provide important clues to reactivate antitumor immune responses suppressed by HPV and to develop effective prognostic and therapeutic tools that may be employed in the treatment of HNSCC.